Method of isolating stem cells

ABSTRACT

The present invention relates, in general, to stem cells, and in particular, to a method of isolating stem cells and to reagents suitable for use in such a method. The invention further relates to stem cell populations isolatable in accordance with the present method.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/632,377, filed Aug. 1, 2003, which is a divisional of U.S.application Ser. No. 09/701,413, filed Feb. 20, 2001, which claimspriority to the National Phase Entry of PCT/US99/28769, filed Dec. 7,1999, which claims the benefit of U.S. Provisional Application No.60/111,195, filed Dec. 7, 1998, all of which are hereby incorporatedherein in their entirety by reference.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The research underlying this invention was supported in part with fundsfrom the National Institutes of Health grant no. CA16783. The UnitedStates Government may have an interest in the subject matter of thisinvention.

TECHNICAL FIELD

The present invention relates, in general, to stem cells, and inparticular to a method of isolating stem cells and to reagents suitablefor use in such a method. The invention further relates to stem cellpopulations isolatable in accordance with the present method.

BACKGROUND OF THE INVENTION

The most primitive hematopoietic stem cells (HSC) will reconstitute allof the hematopoietic lineages for an entire lifespan. These pluripotenthematopoietic stem cells (PHSC) are the transplantable cells that areultimately the targets for gene delivery in stem cell-based genetherapies. One defining characteristic for PHSC is that they willsurvive most cytoablative conditioning regimens. The mechanisms fortheir resistance to these toxic agents suggest potential strategies bywhich these cells can be selected in vitro. One mechanism for drugresistance lies in the ability to efflux toxic substances out of thecell via the multiple drug resistance (MDR) pump. Fluorescent substratesfor the MDR pump have permitted the isolation of PHSC based on theirhigh capacity for dye efflux in a variety of assay systems. Drugresistances may also be conferred by more specific mechanisms. Forexample, a cytosolic aldehyde dehydrogenase (ALDH) mediates resistanceto cyclophosphamide (CPA), an alkylating agent used in cytoreductiveregimens in preparation for bone marrow transplant. Thus, expression ofALDH can be considered a selectable marker for true PHSC.

The therapeutic effectiveness of CPA has been attributed largely to theability of PHSC and intestinal crypt cells to survive the drug regimen.Human hematopoietic progenitors express a cytosolic ALDH and primitivehuman HSC derived from mobilized peripheral blood stem cells can beselected when placed in culture with cyclophosphamide for 7 days. Joneset al. have demonstrated that long-term reconstituting murine PHSC canbe isolated by providing a membrane-permeable fluorescent substrate forALDH and by then selecting cells with the highest levels of ALDHactivity (Jones (1995) Blood 85:2742; Jones et al. (1996) Blood 88:487).In these studies, dansyl aminoacetaldyde (DAAA) was used to stain murinebone marrow cells prepared by countercurrent elutriation.

Preliminary studies using DAAA indicate that this reagent is unusable onpreparations of human hematopoietic cells because the signal intensityof the reagent is too high to resolve discrete cell populations by flowcytometry. The present invention provides a fluorescent ALDH substratethat is free of the problems associated with DAAA and that can be usedin the purification of primitive human hematopoietic cells.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to a novel reagent and method forisolating stem cells, including human stem cells. The reagent is afluorescent substrate for ALDH. The method comprises staining a cellpopulation that includes primitive stem cells with the substrate in thepresence of an inhibitor of MDR activity. ALDH present in the cellsconverts the substrate to a product that is trapped within the cells.Since primitive stem cells have higher levels of ALDH activity thanother cell types, these cells stain brighter than other cell types. Thepresence of the MDR inhibitor reduces the efflux of the convertedsubstrate from the stem cells.

Objects and advantages of the present invention will be clear from thedescription that follows.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

Having thus described the invention in general terms, reference will nowbe made to the accompanying drawings, which are not necessarily drawn toscale, and wherein:

FIGS. 1A-1D. BAAA staining identifies cells with high levels of ALDHactivity. L1210/cpa is a derivative of the L1210 leukemic cell line thatoverexpresses ALDH. (FIGS. 1A and 1B represent L1210 cells, plus DEABand minus DEAB, respectively; FIGS. 1C and 1D represent L1210/cpa cells,plus and minus DEAB, respectively).

FIGS. 2A-2D. Converted BAAA is effluxed by an MDR pump fromhematopoietic cells, particularly primitive CD34+ cells, as evidenced bythe difference between the CD34+ cells that are BODIPY^(bright) (FIG.2B) in the presence and absence of the MDR inhibitor, verapamil (FIGS.2A and 2B are at t=0, minus and plus verapamil, respectively; FIGS. 2Cand 2D are at t=30′, minus and plus verapamil, respectively.)

FIGS. 3A-3D. ALDH^(br) cells (i.e., cells with low SSC properties thatstain brightly with BAAA in the presence of an MDR inhibitor) areenriched for cells with the primitive CD34⁺CD38^(lo/−) immunophenotypetraditionally associated with primitive stem cells.

FIGS. 4A and 4B. The staining intensity with BAAA correlates inverselywith CD38 (FIG. 4A) and CD71 (FIG. 4B) expression in CD34⁺ cells.

FIGS. 5A-5C. ALDH^(br) cells are enriched for early progenitorsequivalent to CD34⁺ cells and are more enriched for very primitiveprogenitors than CD34⁺ cells. (FIG. 5A=progenitors (HPCA), FIG. 5B=earlyprogenitors (5 week LTC) and FIG. 5C=primitive progenitors (8 weekLTC)).

FIG. 6. Preparation of BODIPY-aminoacetaldehyde diethylacetal. Using anamber vial, a solution of aminoacetaldehyde diethyl acetal (0.019 mmol,Aldrich Chemical Co.) in dry tetrahydrofuran (THF, 0.5 mL) was addeddropwise to a solution of BODIPY FL, SE (0.013 mmol, Molecular Probes)in dry THF (0.5 mL). Upon complete addition, the vial was capped and thereaction mixture was stirred for 30 min. The THF was evaporated and theresidue was dissolved in minimal methylene chloride and thenchromatographed on silica gel using ethyl acetate—hexane (1:1) aseluent. The product, BODIPY-aminoacetaldehyde diethylacetal wasrecovered in quantitative yield and identified by proton NMR.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of isolating stem cells and toa reagent suitable for use in such a method. The method comprisescontacting a population of cells comprising stem cells with a detectablesubstrate for aldehyde dehydrogenase (ALDH), which substrate isconverted to a detectable product by ALDH, that product being retainedin the cells. In a preferred embodiment, the substrate isBODIPY-aminoacetaldehyde (BAAA) and efflux of converted BAAA from thecells, particularly the stem cells present in the population, isinhibited by the concurrent use of a MDR-inhibitor.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

The present invention relates to a method of isolating stem cells and toa reagent suitable for use in such a method. The method comprisescontacting a population of cells comprising stem cells with a detectablesubstrate for aldehyde dehydrogenase (ALDH), which substrate isconverted to a detectable product by ALDH, that product being retainedin the cells. In a preferred embodiment, the substrate isBODIPY-aminoacetaldehyde (BAAA) and efflux of converted BAAA from thecells, particularly the stem cells present in the population, isinhibited by the concurrent use of a MDR-inhibitor.

Sources of cell populations that are suitable for use in the presentinvention include umbilical cord blood, bone marrow, peripheral bloodand fetal liver. Any cell population that includes stem cells can beused regardless of tissue origin (e.g., gut, skin muscle, nerve, etc.).While the present method can be expected to be applicable to a varietyof non-human mammalian cell populations, it is particularly useful inisolating human stem cells from sources including those referencedabove.

Substrates suitable for use in the present invention include substratesfor ALDH, particularly specific substrates for ALDH, that are detectableor bear a detectable label and that are converted, by the action of ALDHto products that are detectable or bear the detectable label whichproducts are retained in the cells, particularly, the stem cells. In apreferred embodiment, the substrate is a fluorescent substrate that hasa discrete fluorescence emission profile identical to FITC. An exampleof such a substrate is BAAA.

The optimum amount of substrate to be added to the cell population canreadily be determined by one skilled in the art (see Example). In thecase of BAAA, concentrations can vary, for example, concentrations ofabout 1 μM to 5 μM can be used.

A concentrated solution of the substrate can be added directly to mediumcomprising the cells to be stained or harvested cells can be suspendedin a substrate-containing medium.

In order to inhibit efflux of the converted substrate of the inventionfrom the cells, concurrent use of an inhibitor of MDR is preferred. Anyof a variety of MDR inhibitors can be used, including verapamil. Theinhibitor can be added to the cells simultaneously with the substrate orprior to the addition of the substrate. The optimum amount ofMDR-inhibitor to be used can be readily determined (e.g., by monitoringloss of staining). In the case of verapamil, concentrations can vary,for example, a concentration of about 50 μM can be used.

After exposure of the cell population to the substrate (and the MDRinhibitor) (e.g., about 30 minutes after), those cells that containhigher concentrations of labeled product can be separated from thosethat contain lower concentrations. In the case of the use of afluorescent label, fluorescence activated cell sorting techniques can beused. Stem cells can be purified from other cells of the startingpopulation based on low orthogonal light scattering on a flow cytometer(identifies small cells, like lymphocytes) and/or brightness offluorescence. As shown in the Example that follows, sorting thebrightest 1% of cells yielded a nearly 40-fold enrichment for cells thatinitiate long term cultures. The cell preparations that were recoveredwere up to 65% CD34⁺ cells, most of which were CD38^(−/dim) CD71^(−dim).The invention includes within its scope cell preparations that aregreater than 50% CD34⁺ cells, preferably greater than 75% CD34⁺ cells,more preferably greater than 90% CD34⁺ cells.

The stem cells isolated in accordance with the present invention haveapplication in a variety of therapies and diagnostic regimens. They aresuitable for both transplantation and gene therapy purposes. Forexample, isolation of stem cells from bone marrow or peripheral blood ofpatients with cancer can provide for the separation of stem cells fromcancer cells. In patients undergoing autologous transplantation, suchseparation can be used to reduce the chance that cancer cells arereturned to the patient. Purified autologous stem cells can be ex vivoexpanded to hasten neutrophil, erythroid, and platelet engraftment afterautologous transplantation. Ex vivo expansion can be effected by growthin defined cytokines, on stromal layers, and/or in bioreactors (Emersonet al. (1996) Blood 87:3082). In addition, the incidence of graftfailure can be reduced. This is beneficial for cancer patientsundergoing autologous transplantation, for patients suffering fromauto-immune disorders, and for patients undergoing gene therapy.

Gene therapy approaches involving the present cells involve, in oneembodiment, isolation of autologous stem cells, exposure of the isolatedcells to a gene delivery vector and re-infusion of the modified cellsinto the patient (Smith, J. Hematother. 1:155 (1992)). This approach caninvolve ex vivo culture or the use of vectors capable of transferringgenes into non-dividing cells, thereby rendering ex vivo cultureunnecessary. Gene therapy can be useful in treating, for example,congenital diseases, such as sickle cell anemia, in which case themutant β-globin gene is replaced or supplemented with either the wildtype globin gene or an anti-sickling globin gene. In the treatment ofcancer, drug resistance genes can be introduced into the stem cells toconfer resistance to cytotoxic drugs. This can reduce the incidence andseverity of myelosupporession. For the treatment of infectious diseases,including HIV, anti-viral genes can be introduced into the stem cells sothat they are rendered resistant to the virus (Gilboa and Smith, Trendsin Genetics 10:139 (1994)).

Isolation of stem cells results in the elimination of T-cells that causeGvHD. This elimination can be expected to reduce the incidence andseverity of GvHD in recipients of allogeneic transplants.

Purified allogeneic stem cells can be ex vivo expanded to hastenneutrophil, erythroid, and platelet engraftment after allogeneictransplantation. In addition, the incidence of graft failure can bereduced. This is likely to be particularly important for recipients ofumbilical cord blood transplants, where small cell doses limit thesuccess of transplantation.

Successful engraftment with stem cells can also be expected to inducetolerance.

Such would clearly enhance solid organ transplantation.

It will be appreciated that cells of the present invention can be usedas sources of new genes (e.g., for cytokines and cytokine receptors),including genes important in growth and development.

In addition to their application in treatment and diagnosis strategies,the stem cells of the invention can be used in screening protocols toidentify agents that can be used, for example, to promotedifferentiation or growth and/or engraftment of hematopoietic cells. Inone such protocol, stem cells are contacted with a test compoundsuspected of inducing differentiation and the ability of the testcompound to effect differentiation determined (using, for example,microscopic and flow cytometric examination). In another screeningprotocol, stem cells are contacted with a test compound suspected ofinducing proliferation and/or engraftment and the ability of the testcompound to effect proliferation and/or engraftment determined using invitro long term colony assays or in vivo immunodeficient mice models (egSCID NOD mice). (See Peault et al. (1993) Leukemia 7:s98-101).

In addition to the above, the substrate of the invention can be used toidentify tumors that may be resistant to cyclophosphamide via upregulation of ALDH activity.

In accordance with this embodiment, cells of the tumor can be contactedwith the detectable substrate, e.g., BAAA, and MDR inhibitor underconditions such that the substrate enters the cells and is convertedtherein to the detectable product. Cells that stain brightly (e.g., withBAAA) can be expected to be cyclophosphamide resistant.

The invention also relates to kits that can be used to prepare the cellsof the invention. The kits can comprise reagents (e.g., ALDH substrate)that can be used to effect isolation of the stem cells. In a preferredembodiment, the kit includes BODIPY aminoacetaldehyde diethyl acetaldisposed within a container means. The kit can also include, disposedwithin a container means, an MDR inhibitor, such as verapamil.

Certain aspects of the present invention are described in greater detailin the non-limiting Examples that follow.

EXPERIMENTAL Example 1

Preparation of BODIPY amino acetaldehyde diethyl acetal

The aldehyde dehydrogenase substrate is prepared as BODIPYaminoacetaldehyde diethyl acetal and lyophilized in 0.5 micromolealiquots. These preparations are stable indefinitely when stored at −20°C. The acetal is then solubilized in DMSO to a final concentration of 5mM. This solution has been found to be stable at 4° C. for up to 1 week.To convert the acetal to an acetaldehyde, aliquots of this solution arebrought to a final concentration of 1 N HCl. Under these conditions theacetal has a half life of 15 minutes. After 2 hours in 1 N HCl, the vastmajority of the BODIPY aminoacetaldehyde diethyl acetal has converted toBODIPY aminoacetaldehyde (BAAA), and is then diluted to 200-250 mM inDulbecco's phosphate buffered saline (PBS). This stock is added directlyto cells prepared in Iscove's Modified Dulbecco's Medium (IMDM) with 2%FCS at concentrations ranging from 1 to 5 μM.)(See also FIG. 6.).

Antibody Reagents.

Directly-conjugated fluorescent antibodies directed against CD2 (Leu5;FITC), CD3 (Leu4; PerCP), CD5 (Leu1; PE), CD7 (Leu9; FITC), CD10 (CALLA;FITC), CD11b (Leu15; PE), CD14 (Leu M3; PE), CD19 (Leu12; FITC), CD33(LeuM9; PE), CD34 (HPCA2; FITC and PE), CD38 (Leu17; PE), CD56 (Leu19;PE) and HLA-DR (FITC) from Becton Dickinson Immunocytometry Systems(BDIS; San Jose, Calif.) were used. Anti-CD7 (3A1; PE) and anti-CD45(KC56; PE) were purchased from Coulter Corporation (Hialeah, Fla.);anti-CD3 (UCHT1; PE), anti-CD16 (3G8; PE), anti-CD19 (J4.119; PE) aswell as the pooled anti-CD34 antibodies (QBEnd10, Immu-133, Immu-134;PE) from Immunotech, Inc. (Westbrook, Me.); anti-CD3 (B-B 11; FITC) andCD38 (B-A6; FITC) from BioSource International (Camarillo, Calif.);anti-CD45RA (F8-11-13; PE) from Southern Biotechnology Associates, Inc.(Birmingham, Ala.); and anti-CDw90 (5E10; PE) from PharMingen, Inc. (SanDiego, Calif.).

Cell Lines.

K562, L1210 and L1210/cpa cells (ATCC) were maintained in suspension inRPMI 1640 media supplemented with 10% Fetal Calf Serum (FCS) and 5×10⁻⁵M β-mercaptoethanol.

Preparation of Human Umbilical Cord Blood.

Human umbilical cord blood (UCB), intended for disposal, was collectedinto sterile bottles containing anticoagulant citrate buffer. The UCBused in these studies were processed within 24 hours of being harvested.White cells were enriched through a preliminary red cell agglutinationwhere the UCB was diluted 1:2 with Dulbecco's phosphate buffered saline(PBS) at room temperature. These cells were then brought to a finalconcentration of 1% Hespan (DuPont Pharma, Wilmington, Del.) and wereleft to stand undisturbed for 1 hour. Non-agglutinated white blood cellswere harvested and residual red cells were hemolysed at 37° C. in 0.17 MNH₄Cl containing 10 mM Tris-HCl, pH 7.2 and 200 mM EDTA. The recoveredcells were washed in IMDM containing 2% FCS and mononuclear cells arethen purified using Ficoll-Hypaque (1.077 g/ml). When held overnight,the cells were kept on ice in a 4° C. refrigerated room in IMDM with 20%FCS.

Cell Staining and Fluorescence-Activated Cell Sorting.

Mononuclear UCB cells were resuspended at 10⁶ cells/ml in IMDMcontaining 2% FCS and were labeled with 1 μM BAAA for 30 min. When used,verapamil was included at 50 mM. After staining, the cells were washedwith ice cold staining media and maintained on ice until their analysisand sorting. The cells were then resuspended in staining media with 10mg/ml 7-aminoactinomycin D (7AAD) (Molecular Probes; Eugene, Oreg.). Forantibody staining to permit multiparameter analyses, the cells wereresuspended in staining media (100 μl) and antibodies were addeddirectly to the cell suspensions. The cells were incubated on ice for 20min. and then washed again in ice cold staining media. The cells werethen analyzed or sorted on a FACStar Plus cell sorter (BDIS) equippedwith dual Coherent I-90 lasers+an argon-dye laser. The BAAA was excitedat 488 nm and emissions were detected using 515 DF20 filter in FL1. Deadand dying cells were excluded on the basis of their high emission in thefar red wavelength due to their uptake of 7AAD.

For analyses of cell surface antigens on cells previously sorted basedon BAAA staining, the cells were pelleted and resuspended in IMDM with2% FCS. The cells were then held at 37° C. for 1-2 hours to permitefflux. The cells were then pelleted and fluorescence-conjugatedantibodies were added directly to the cells. Following incubations for20 minutes, the cells were washed with PBS/2% FCS and were fixed in 1%formaldehyde in PBS/2% FCS. In all surface marker analyses, nodifferences were noted between analyses with cells stainedsimultaneously with BAAA and with antibodies and those analysesperformed on FACS® sorted cells that were subsequently stained withantibodies.

Hematopoietic Progenitor Colony Assays and Long Term Cultures.

ALDH^(br) cells were isolated directly from mononuclear UCB cells whichhad been stained with BAAA. For these assays, the ALDH^(br) was definedas 1% of the lymphocyte gate of the UCB.

Hematopoietic progenitor colony assays were performed by plating 100-200cells in MethoCult H4431 containing agar leukocyte conditioned media andrecombinant human erythropoietin (StemCell Technologies, Inc.). Thecells were incubated in a humidified chamber at 37° C. with 5% CO₂.Hematopoietic colonies (>100 cells) were then scored at 14 to 18 daysafter initiating the cultures. Long term cultures were maintained onstromal layers of murine MS-5 cells (provided by Dr. Tadashi Sudo of theKirin Pharmaceutical Research Laboratory, Gunma, Japan) (Issaad, Blood81:2916 (1993)). MS-5 stromal cells were seeded into 24-well plates(Corning Costar Corp., Cambridge, Mass.) at 5×10⁴ cells/well in DMEMsupplemented with 10% FCS and cultured at 37° C. When the monolayersapproached 80% confluence they were γ-irradiated from a cesium source(40 Gy). After irradiation, fresh media was provided to the cultures.For the MS-5 cells, the culture media was replaced entirely with MEMαsupplemented with 10% FCS, 10% equine serum, β-mercaptoethanol,pyruvate. Long term cultures were initiated with 400-2000 hematopoieticprogenitor cells/well and were maintained at 33° C. with 5% CO₂. Atweekly intervals half the media from each well was removed so that themedia could be replenished. Adherent and non-adherent cells wereharvested after 5 or 8 weeks and plated into HPC assays as describedabove. As shown in the Example that follows, sorting the brightest 1% ofcells yields a nearly 40-fold enrichment for cells that initiate longterm cultures. The cell preparations that were recovered were up to 65%CD34⁺ cells, most of which were CD34⁺ cells, most of which wereCD38^(−/dim) CD71^(−dim).

Results

Synthesis of BODIPY aminoacetaldehyde diethyl acetal.

Due to the inherent instability of aldehydes in aqueous solution, thereagent is prepared and stored as an acetal. Immediately prior to itsuse, the acetal is converted to an aldehyde in 1 N HCL. the aldehyde isfreely soluble in PBS and can be added directly to cells prepared inIMDM with 2% fetal calf serum at 106 cells per ml. As anaminoacetaldehyde, the reagent is membrane permeable; however, in thepresence of the aldehyde dehydrogenase (ALDH), the aldehyde moiety isconverted to a carboxylic acid that is retained in the cell.Intracellular fluorescence can be used to select cells.

Converted BAAA is a Specific Substrate for ALDH.

To assay whether BAAA would permit the specific selection of ALDH⁺cells, studies initially determined an optimal response dose for theBAAA reagent in a murine cell line previously selected forcylophoshamide-resistance, L1210/cpa, that is known to be ALDH⁺ (FIG.1). The parental cell line, L1210 (FIGS. 1A and 1B) iscylophosphamide-sensitive and ALDH⁻. This cell line exhibitedessentially no response to BAAA. In addition, a potent inhibitor ofALDH, diethylbenzaldehyde (DEAB), was used to demonstrate thespecificity of the BAAA signal. A 10-fold molar excess of DEAB totallyblocked the fluorescent response (FIG. 1C). Therefore, BAAA was able todetect ALDH⁺ cells. In these studies, the BAAA could be used at a finalconcentration as low as 5 μM. This molar concentration is 10-fold lowerthan that used with the dansylated reagent.

Multiple different ALDH isoenzymes exist and these may display differentabilities to convert BAAA. It has been suggested that resistance tocyclophosphamide is primarily mediated by a specific ALDH isoenzyme,ALDH1. Therefore, a human cell line known to express ALDH1, K562, wasassayed with this novel reagent. K562 cells converted BAAA and werepositive for ALDH in these assays. This response was entirely inhibitedby DEAB. Thus, BAAA can serve as a specific substrate for human ALDH1and can be used to identify primary human cells that demonstrateresistance to cyclophosphamide.

Primary UCB Cell Preparations Contain Subsets of ALDH^(br) Cells.

Having demonstrated the effectiveness of this reagent on continuous celllines, BAAA was assayed on primary human cells. Umbilical Cord Blood(UCB) was chosen for its increasing promise as a source fortransplantable hematopoietic stem cells. For these studies, the UCB wasunfractionated except for having been prepared for mononuclear cellsover Ficoll-Hypaque. This separation is significant in that two matureALDH⁺ cell types, erythrocytes and megakaryocytes, are removed. The BAAAwas tested on UCB cells prepared in IMDM with 2% FCS at 10⁶ cells/ml(FIG. 2). The UCB cells were very responsive to the BODIPY reagent, andappeared to be much more sensitive than the continuous cell lines hadbeen. The BAAA was therefore titrated to an optimal concentration of 1μM. This was the best concentration for resolving ALDH^(br)subpopulations. The response was inhibited in the presence of excessDEAB and was therefore specific for ALDH. This molar concentration is50-fold lower than the concentration of dansyl aminoacetaldehyde thathad previously been used to detect murine pluripotent hematopoietic stemcells.

The fluorescence emission from BAAA-stained UCB cells exhibited abimodal response. The brighter peak of fluorescence emission wasattributed to mature monocytes, suggesting monocytes express a uniformlevel of ALDH. Hematopoietic stem cells are small, non-complex cells.Indeed, murine ALDH⁺ PHSC were first enriched using countercurrentelutriation. Therefore, the BODIPY signal was examined only innon-complex cells that exhibited low inherent orthogonal lightscattering (SSC^(lo)) (FIG. 3A). The majority of the SSC^(lo) UCB cellswere ALDH^(neg/dim) (FIG. 1B). This was not unexpected since theSSC^(lo) cells are predominantly lymphocytes, and most lymphocytes donot express ALDH. However, a small, clearly-defined subpopulation of theSSC^(lo) UCB cells was ALDH^(br) (FIG. 3A).

BODIPY Aminoacetate is a Substrate for the MDR Efflux Pump.

In addition to expressing ALDH, PHSC should also express high levels ofthe P-glycoprotein or multiple drug resistance (MDR) efflux pump. Sincethis reagent had never been previously characterized, the susceptibilityof converted BAAA to MDR efflux was assayed. Although BODIPYaminoacetaldehyde passes through the cell membrane without activetransport, the product of the ALDH conversion (BODIPY aminoacetate)might well be a substrate for the MDR pump. To investigate thispossibility, UCB cells were stained with BAAA in the presence of 50 μMverapamil, a competitive inhibitor of the MDR efflux pump. Theverapamil-treated cells exhibited a consistently-higher fluorescencewhen compared with BAAA-stained cells that had not been simultaneouslytreated with verapamil (FIG. 2). A substantial population of ALDH^(dim)cells were effected by the verapamil treatment. Most importantly, thepercentage of ALDH^(br) cells increased by 1.8 fold in the presence ofverapamil. In verapamil-treated cells, the ALDH^(br) subpopulation wasequivalent to 0.8±% of the SSC^(lo) cells. In contrast, in cellpreparations that received no verapamil, the same fluorescence intensityrepresented only 0.46±% of the SSC^(lo) cells. This indicated that theALDH^(br) SSC^(lo) UCB cells retain the converted BAAA more effectivelyif the efflux activity of the MDR pump is inhibited.

ALDH^(br) SSC^(lo) UCB Cells are Highly Enriched for Primitive CD34⁺Cells.

With verapamil treatment, the ALDH^(br) SSC^(lo) UCB cells containedalmost 90% CD34⁺ cells, indicating that at least some hematopoieticprogenitors are present (FIG. 3D). However, CD34 is expressed by a broadrange of hematopoietic progenitors that includes lineage committed cellsas well as pluripotent progenitors. Therefore, the developmentalpotential of the ALDH^(br) SSC^(lo) UCB cells was analyzed. Initially,the immunophenotype of these cells was more carefully defined. Theimmunophenotype would in no way be conclusive; however, theprimitiveness of the cell population could be inferred by examining twoactivation markers that are typically associated with thedifferentiation of primitive cells to more lineage-committedhematopoietic cells, CD38 and CD71. The most primitive subsets of CD34⁺cells have little to no expression of the activation antigens CD38 orCD71. In UCB cell preparations with BAAA and with antibodies specificfor CD34 and CD38, the ALDH^(br) SSC^(lo) UCB cells provided asingle-step enrichment for essentially purified CD34^(br) CD38^(dim)cells. Furthermore, when CD34⁺ UCB cells were examined independently,ALDH expression was inversely proportional to the expression of bothCD38 and CD71 (FIGS. 4A and 4B). Thus, the ALDH^(br) SSC^(lo) UCB cellsappear to contain the primitive CD34⁺ cells as defined byimmunophenotype.

To assay the developmental potential of the ALDH^(br) SSC^(lo) UCBcells, these cells were isolated and placed into both short-term andlong-term assays for myeloerythroid progenitors. The short-term assayused, the hematopoietic progenitor colony assay (HPCA), quantifieslineage committed cells at the time of the initial isolation. Moreprimitive progenitors were also assayed by maintaining the ALDH^(br)SSC^(lo) UCB cells on stroma for either 5 or 8 weeks prior to performingthe HPCA (FIG. 5).

Results:

HPCA—ALDH^(br) SSC^(lo) essentially equivalent to CD34⁺ cells.

LTC —5 wk—ALDH^(br) SSC^(lo) essentially equivalent to CD34⁺ cells.

LTC =8 wk—ALDH^(br) SSC^(lo) outperforms CD34⁺ cells.

Example 2

ALDH^(br) UCB cells have been shown to be predominantly CD34⁺CD38^(−/lo)and highly enriched for early myeloid progenitors. The current study wasundertaken to determine whether the ALDH^(br) CD34⁺ UCB cells wereenriched for lymphoid progenitors as well. In three experiments,cultures of ALDH^(br) CD34 UCB cells were established on AFT024 stromalcells in the presence of Kit ligand, Flt3 ligand, IL-3 (1st day only),IL-2 and IL-7 at various dilutions. After 7-8 weeks, the cultures wereanalyzed for lymphocyte growth as determined by expression of CD56,CD10, CD19 or CD20. TABLE 1 ALDH^(br) total wells wells with lymphocyte⁺cells/well initiated viable cells wells 1000 6 5 5 250 16 12 12 62 48 4040 16 48 34 34 10 24 20 17

The AFT024 cultures primarily favored the growth of presumptive NKcells, so to more effectively test whether ALDH^(br) CD34⁺ UCB cellscontained B-lymphoid progenitors, they were cultured on the W20 stromalcell line supplemented with the same cytokine combination. Of 12cultures established with 100 ALDH^(br) CD34⁺ cells, all produced CD56⁺and CD10⁺ cells at nearly equivalent proportions. 2 of the 12 wells alsocontained CD19⁺ cells.

In summary, the ALDH^(br) CD34⁺ UCB population appears to be highlyenriched for both myeloid and lymphoid hematopoietic progenitors.

All documents cited above are hereby incorporated in their entirety byreference. One skilled in the art will appreciate from a reading of thisdisclosure that various changes in form and detail can be made withoutdeparting from the true scope of the invention.

1. A stem cell population isolated from a population of cells bycontacting said population of cells with a detectable substrate foraldehyde dehydrogenase (ALDH) under conditions such that ALDH present incells of said population converts said substrate to a detectable productthat is retained by said cells present in said population, wherein saidstem cells have a higher concentration of said detectable product thannon-stem cells present in said population, and separating said stemcells from said non-stem cells of said population on the basis of saidhigher concentration of detectable product wherein said substrate isBODIPY aminoacetaldehyde (BAAA).
 2. The stem cell population accordingto claim 1 wherein said population of cells is a human population ofcells.
 3. The stem cell population of claim 2, wherein said populationof cells is from a source selected from the group consisting ofumbilical cord blood, bone marrow, peripheral blood and fetal liver. 4.The method of claim 3, wherein said population of cells is fromumbilical cord blood.
 5. The method of claim 3, wherein said populationof cells is from bone marrow.
 6. The method of claim 3, wherein saidpopulation of cells is from peripheral blood.
 7. The method of claim 3,wherein said population of cells is from fetal liver.